一株产酸性蛋白酶菌株的筛选、鉴定及发酵条件优化
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摘要
化肥过度使用导致比较严重的耕地退化问题,极大影响了作物的产量与品质。本研究筛选产酸性蛋白酶菌株,结合现代发酵技术,尝试开发一种新型氨基酸生物有机肥。首先利用透明水解圈法和福林酚法,从鱼粉水解液中筛选得到一株产酸性蛋白酶菌株Zju-c,对其进行形态学观察及26S rRNA分子鉴定,并对鱼粉菌解条件和培养基成分进行部分优化。经生理生化分析发现,该菌株可直接利用大多数糖及少部分醇,能水解酪素、淀粉、明胶以及酒石酸盐,生长速度较快,可耐受13%NaCl;通过BLAST比对,确定为毕赤酵母属Pichia galeiformis (GenBank No. JF781422.1)。最佳发酵培养基成分为300 g/L鱼粉、50 g/L葡萄糖、30 g/L大豆蛋白胨、30 g/L NaCl和0.9 mg/mL Mg2+。在30℃、起始pH 5、3%接种量以及180 r/min的优化发酵条件下振荡培养7 d,以游离氨基酸含量和酶活为发酵水平衡量指标,发酵液中氨基酸含量由最初的29.4±2.1 mg/mL提高至88.4±1.4 mg/mL,酶活由190.2±2.9 U/mL提高到400.1±3.8 U/mL。上述研究结果提示,该菌株在利用发酵鱼粉研制液体生物有机肥中具有较好的应用价值。
Excessive use of fertilizers leads to serious degradation of cultivated lands which greatly affects crop yield and quality. Through the screening of acid-protease strains, combined with modern fermentation techniques, the present study attempted to develop new amino acid organic fertilizers. An acid-protease strain named Zju-c was screened from the hydrolytic liquid of fish meal using the transparent hydrolysis circle method and the Folin-Phenol method. Morphological observation and 26 S rRNA molecular identification were performed, and the fermentation condition was optimized. Results of physiological and biochemical reactions showed that most sugars and a small portion of alcohols could be utilized directly, and casein, starch, gelatin,and tartrates could be hydrolyzed, and the growth rate was fast. The concentration of NaCl can be tolerated upto 13%. The BLAST of 26 S r RNA sequence in GenBank indicated that the strain belonged to Pichia pastoris and the phylogenic tree of the strain, which was generated by Vector NTI Suite 7 software based on 26 S rRNA sequence, showed that it was most close to Pichia galeiformis(GenBank No. JF781422.1) strain with 100% ofidentity. In addition, the optimum fermentation conditions were 300 g/L fish meal, 50 g/L glucose, 30 g/L soy peptone, 30 g/L NaCl and 0.9 mg/mL Mg2+with 3% inoculation quantity, with the culture condition of 30 ℃,pH 5.0, and 180 r/min for 7 d.The contents of free amino acids and enzymic activity were the fermentation index, total amino acids content in the fermentation broth eventually increased from initial 29.4±2.1 mg/mL to88.4 ± 1.4 mg/m L, and the enzymic activity was optimized from 190.2 ± 2.9 U/mL to 400.1 ± 3.8 U/mL.Therefore, the strain has good application potential for the development of liquid bio-organic fertilizer.
Excessive use of fertilizers leads to serious degradation of cultivated lands which greatly affects crop yield and quality. Through the screening of acid-protease strains, combined with modern fermentation techniques, the present study attempted to develop new amino acid organic fertilizers. An acid-protease strain named Zju-c was screened from the hydrolytic liquid of fish meal using the transparent hydrolysis circle method and the Folin-Phenol method. Morphological observation and 26 S rRNA molecular identification were performed, and the fermentation condition was optimized. Results of physiological and biochemical reactions showed that most sugars and a small portion of alcohols could be utilized directly, and casein, starch, gelatin,and tartrates could be hydrolyzed, and the growth rate was fast. The concentration of NaCl can be tolerated upto 13%. The BLAST of 26 S r RNA sequence in GenBank indicated that the strain belonged to Pichia pastoris and the phylogenic tree of the strain, which was generated by Vector NTI Suite 7 software based on 26 S rRNA sequence, showed that it was most close to Pichia galeiformis(GenBank No. JF781422.1) strain with 100% ofidentity. In addition, the optimum fermentation conditions were 300 g/L fish meal, 50 g/L glucose, 30 g/L soy peptone, 30 g/L NaCl and 0.9 mg/mL Mg2+with 3% inoculation quantity, with the culture condition of 30 ℃,pH 5.0, and 180 r/min for 7 d.The contents of free amino acids and enzymic activity were the fermentation index, total amino acids content in the fermentation broth eventually increased from initial 29.4±2.1 mg/mL to88.4 ± 1.4 mg/m L, and the enzymic activity was optimized from 190.2 ± 2.9 U/mL to 400.1 ± 3.8 U/mL.Therefore, the strain has good application potential for the development of liquid bio-organic fertilizer.
引文
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钱存柔,黄仪秀.2000.微生物学实验教程[M].北京:北京大学出版社,pp.99-100.(Qian C R,Huang Y X.2000.Microbiology Experiment[M].Beijing University Press,Beijing,China,pp.99-100.)
王进.2013.利用复合微生物菌剂制备浒苔生物有机肥及其对作物生长影响的研究[D].硕士学位论文,中国海洋大学,导师:王鹏,pp.4-10.(Wang J.2013.Studies on the production of Enteromorpha proliferabio-organic fertilizer by complex microbial inoculant and itseffect on the growth of crop[D].Thesis for M.S.,China Ocean University,Suppervisor:Wang P,pp.4-10.)
喻曼,沈阿林,许育新等.2015.一种复合氨基酸液肥及其制备方法.中国,CN105175161A[P].(Yu M,Shen A L,Xu Y X,et al.2015.A compound amino acid liquid fertilizer and its preparation method.China,CN105175161A[P].)
赵玉谨,刘为,刘海军等.2015.几株共同发酵水产下脚料菌株的筛选及鉴定[J].上海海洋大学学报,3(24):465-471.(Zhao Y J,Liu W,Liu H J,et al.2015.Screening and identification of several microbes fermenting byproduct of fish processing[J].Journal of Zhejiang Ocean University,3(24):465-471.)
Araujo A S F,Miranda A R L,Oliveira M L J.2015.Soil microbial properties after 5 years of consecutive amendment with composted tannery sludge[J].Environmental Monitoring and Assessment,187(1):202-208.
Chatterjee P M,Datta S,Tiwari D P.2018.Selection of an effective indicator for rapid detection of microorganisms producing gamma-polyglutamic acid and its biosynthesis under submerged fermentation conditions using Bacillus methylotrophicus[J].Applied Biochemistry and Biotechnology,185(1):270-288.
Gondek K,Mierzwa-Hersztek M,Kopec M.2018.Mobility of heavy metals in sandy soil after application of composts produced from maize straw,sewage sludge and biochar[J].Journal of Environmental Management,210:87-95.
Heimersson S,Vanstrom Cederberg C.2017.Improved life cycle modelling of benefits from sewage sludge anaerobic digestion and land application[J].Resource Conservation and Recycling,122(14):126-134.
Kumar S,Stecher G,Tamura K.2016.MEGA7:Molecular Evolutionary genetics analysis version 7.0 for bigger datasets[J].Molecular Biology and Evolutions,33(7):1870-1874.
东秀珠,蔡妙瑛.2001.常见细菌系统鉴定手册[M].北京科学出版社,pp.349-398.(Dong X Z,Cai M Y.2001Manual of identification for general bacteriology[M]Publishing Company of Science,Beijing,China,pp349-398.)
高小文.2014.一种氨基酸液肥的制备方法.中国CN104058801A[P].(Gao X W.2014.A amino acid liquid fertilizer and its preparation method.China CN104058801A[P].)
李静.2008.海洋酵母菌Metschnikowia reukaufii W6b酸性蛋白酶的生产、基因克隆和表达的研究[D].博士学位论文,中国海洋大学,导师:池振明,pp.34-36.(Li J2008.Study on producting,gene cloning and expression of an acid protease from marine yeast Metschnikowia reukaufii W6b[D].Thesis for Ph.D.,China Ocean University,Suppervisor:Chi Z M,pp.34-36.)
李静,李荣,沈其荣等.2017.添加动物源氨基酸水解液研制生物有机肥[J].环境科学研究,30(6):967-973.(Li JLi R,Shen Q R,et al.2017.Development of bio-organic fertilizer by adding amino acids hydrolyzed from animal carcasses[J].Research of Environmental Sciences30(6):967-973.)
钱存柔,黄仪秀.2000.微生物学实验教程[M].北京:北京大学出版社,pp.99-100.(Qian C R,Huang Y X.2000.Microbiology Experiment[M].Beijing University Press,Beijing,China,pp.99-100.)
王进.2013.利用复合微生物菌剂制备浒苔生物有机肥及其对作物生长影响的研究[D].硕士学位论文,中国海洋大学,导师:王鹏,pp.4-10.(Wang J.2013.Studies on the production of Enteromorpha proliferabio-organic fertilizer by complex microbial inoculant and itseffect on the growth of crop[D].Thesis for M.S.,China Ocean University,Suppervisor:Wang P,pp.4-10.)
喻曼,沈阿林,许育新等.2015.一种复合氨基酸液肥及其制备方法.中国,CN105175161A[P].(Yu M,Shen A L,Xu Y X,et al.2015.A compound amino acid liquid fertilizer and its preparation method.China,CN105175161A[P].)
赵玉谨,刘为,刘海军等.2015.几株共同发酵水产下脚料菌株的筛选及鉴定[J].上海海洋大学学报,3(24):465-471.(Zhao Y J,Liu W,Liu H J,et al.2015.Screening and identification of several microbes fermenting byproduct of fish processing[J].Journal of Zhejiang Ocean University,3(24):465-471.)
Araujo A S F,Miranda A R L,Oliveira M L J.2015.Soil microbial properties after 5 years of consecutive amendment with composted tannery sludge[J].Environmental Monitoring and Assessment,187(1):202-208.
Chatterjee P M,Datta S,Tiwari D P.2018.Selection of an effective indicator for rapid detection of microorganisms producing gamma-polyglutamic acid and its biosynthesis under submerged fermentation conditions using Bacillus methylotrophicus[J].Applied Biochemistry and Biotechnology,185(1):270-288.
Gondek K,Mierzwa-Hersztek M,Kopec M.2018.Mobility of heavy metals in sandy soil after application of composts produced from maize straw,sewage sludge and biochar[J].Journal of Environmental Management,210:87-95.
Heimersson S,Vanstrom Cederberg C.2017.Improved life cycle modelling of benefits from sewage sludge anaerobic digestion and land application[J].Resource Conservation and Recycling,122(14):126-134.
Kumar S,Stecher G,Tamura K.2016.MEGA7:Molecular Evolutionary genetics analysis version 7.0 for bigger datasets[J].Molecular Biology and Evolutions,33(7):1870-1874.